Mutations in Extensively Drug‐Resistant Mycobacterium tuberculosis That Do Not Code for Known Drug‐Resistance Mechanisms

Mutations in Extensively Drug Resistant Mycobacteriumtuberculosis that do not Code for Known Drug-ResistanceMechanisms

Alifiya S. Motiwala1, Yang Dai1, Edward C. Jones-López1, Soo-Hee Hwang2, Jong SeokLee3, Sang Nae Cho4, Laura E. Via5, Clifton E. Barry 3rd5, and David Alland1

1Division of Infectious Disease, Department of Medicine, and the Ruy V. Lourenço Center for theStudy of Emerging and Reemerging Pathogens, New Jersey Medical School, University of Medicineand Dentistry of New Jersey, Newark, New Jersey 2National Masan Tuberculosis Hospital, Masan,Republic of Korea 3International Tuberculosis Research Center, Masan, Republic of Korea4Department of Microbiology, Yonsei University College of Medicine, Seoul, Republic of Korea5National Institutes of Health, Bethesda, Maryland

AbstractHighly-lethal outbreaks of multi drug-resistant (MDR) and extensively drug-resistant (XDR)tuberculosis are increasing. Whole-genome sequencing of KwaZulu-Natal MDR and XDR outbreakstrains prevalent in HIV patients by the Broad Institute identified 22 novel mutations which wereunique to the XDR genome or shared only by the MDR and XDR genomes and not already knownto be associated with drug-resistance. We studied the 12 novel mutations which were not located inhighly-repetitive genes to identify mutations that were truly associated with drug-resistance or likelyto confer a specific fitness advantage. None of these mutations could be found in a phylogeneticallyand geographically diverse set of drug–resistant and susceptible M. tuberculosis isolates, suggestingthat these mutations are unique to the KZN clone. Examination of the 600 bp region flanking eachmutation revealed 26 new mutations. We searched for a convergent evolutionary signal in the newmutations for evidence that they emerged under selective pressure, consistent with increased fitness.However, all but one rare mutation were monophyletic, indicating that the mutations were markersof strain-phylogeny rather than fitness or drug-resistance. Our results suggest that virulent XDRtuberculosis in immunocompromised HIV patients can evolve without generalizable fitness changesor other XDR-specific mutations.

KeywordsXDR tuberculosis evolution

IntroductionMulti drug-resistant (MDR) and extensively drug resistant (XDR) Mycobacteriumtuberculosis is an expanding problem in many countries [1]. The high mortality rates associated

Corresponding Author: David Alland, MD, Professor of Medicine and Chief, Division of Infectious Disease, Assistant Dean for ClinicalResearch, New Jersey Medical School – UMDNJ, 185 South Orange Avenue, MSB A920C, Newark, NJ 07103, Tel: (973) 972-2179,Fax: (973) 972-0713, [email protected] authors declare that they do not have a commercial or other association that might pose a conflict of interest.

NIH Public AccessAuthor ManuscriptJ Infect Dis. Author manuscript; available in PMC 2011 March 15.

Published in final edited form as:J Infect Dis. 2010 March 15; 201(6): 881–888. doi:10.1086/650999.

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with some drug-resistant strains are particularly worrisome. The recently described XDRtuberculosis outbreak in Tugela Ferry KwaZulu-Natal, South Africa, had a mortality rate of98% among patients infected with the human immunodeficiency virus (HIV) [2]. It is notknown whether epidemic drug-resistant strains have evolved special mechanisms that facilitatedrug-resistance acquisition, maintain fitness or promote person-to-person transmission.Conventional wisdom has held that M. tuberculosis acquires MDR and XDR through a stepwiseaccumulation of chromosomal mutations that each confers resistance to individual drugs [3].Single-step MDR mutations have not been conclusively identified. Resistance mutations havebeen shown to be associated with a fitness “cost” in many bacteria including M. tuberculosis,where the cost appears to be dependent on specific mutation and strain type [4]. Several invitro studies have suggested that clinical M. tuberculosis has found ways to compensate forthe fitness cost of antibiotic resistance [5]. However, except for the relatively rare occurrenceof ahpC promoter up mutations, no molecular mechanisms that maintain fitness in drug-resistant clinical M. tuberculosis have been identified.

Comparative whole genome sequencing studies have the potential to identify biologicallysignificant mutations in drug-resistant M. tuberculosis strains by providing an unbiased scanof the total genomic changes that accompany resistance acquisition. The Broad Institute hasrecently released the complete genomic sequences of a Tugela Ferry XDR outbreak isolate andtwo related MDR and drug susceptible (DS) isolates [6,7]. Comparing the three Tugela Ferrygenomes to a representative DS isolate (strain F11) from the same geographic region revealed15 mutations that were unique to the Tugela Ferry XDR isolate (XDR-specific mutations), and18 mutations that were shared by both the Tugela Ferry XDR and MDR isolates but were notpresent in the DS isolate (XDR/MDR-specific mutations). As expected, many of thesemutations mapped to known drug-resistance targets and/or had previously known associationswith drug resistance. However, 22 mutations were completely novel and did not occur in genesthat had been described previously in association with drug resistance. It is tempting to assumethat these novel mutations represent unidentified drug resistance mutations, new mutationsrelevant to XDR evolution or fitness adaptations.

Mutations that are highly selected during MDR and XDR evolution should not be restricted tothe Tugela Ferry XDR strain or its related MDR isolate. Rather, these biologically importantmutations should occur with detectible frequencies in unrelated MDR and XDR M.tuberculosis strains from around the world. This hypothesis is supported by the observationthat the common mutations that are known to cause resistance to the first line anti-tuberculosisdrugs isoniazid, ethambutol [8], and rifampin (data not shown) can be found in different andunrelated populations, reflecting convergent evolution.

MethodsIdentification of mutations specific to the extensively drug resistant (XDR) or both the XDRand multi drug resistant (MDR) isolates sequenced from Tugela Ferry KwaZulu-Natal, SouthAfrica

Polymorphisms unique to the XDR strain or shared by the XDR and MDR strains were obtainedfrom the publicly available comparative analysis of the XDR (KZN 605), MDR (KZN 1435),and drug-sensitive (DS) (KZN 4207) strains published by the Microbial Sequencing Center atthe Broad Institute [7].

Selection of clinical M. tuberculosis isolates for the studyA total of 35 drug-resistant and 34 pan-susceptible isolates were selected from a collection ofclinical M. tuberculosis isolates obtained from reference laboratories or major medical centersin Afghanistan (n=1), Australia (n=1), China (n=1), Colombia (n=14), India (n=6), Korea

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(n=1), Mexico (n=10), New York City (n=6), Philippines (n=1), Somalia (n=1), Spain (n=3),Sudan (n=1), Texas (n=22), and Vietnam (n=1) [9,10]. Effort was made to include multi-drugresistant isolates where possible. Each of the isolates was assigned to one of 10 SNP clustergroup/sub-group using nine SNP markers developed for this purpose [11] and then mappedonto the previously described phylogenetic tree [12] (Fig. 2). Additional six extensively drug-resistant isolates were obtained Mulago Hospital, Uganda (n=1), and National MasanTuberculosis Hospital, Korea (n=5). All isolates were subjected to susceptibility testing asdescribed previously [10].

Detection of mutations in M. tuberculosis study isolatesPrimers were designed to amplify an approximately 600bp region flanking the mutations ofinterest per the Mycobacterium tuberculosis H37Rv genome sequence (GenBank AccessionNC_000962). Additionally, the 1357bp Rv3616c-Rv3617 intergenic region was sequenced inall isolates. The entire length of each gene identified by the Tugela Ferry MDR-XDR mutationswas sequenced in the six XDR isolates. PCR amplification was performed using Hotstar Taqpolymerase (Qiagen Inc.). The PCR products were sequenced using standard dye terminatorchemistry and analyzed on an automated DNA sequencer (3700 DNA Analyzer, AppliedBiosystems). All mutations were confirmed by sequencing the reverse strand, except whenthey were abundant, in which case a subset of isolates were retested for each mutation.

ResultsWe tested for mutations that were shared by the Tugela Ferry strain and unrelated M.tuberculosis strains by looking for the Tugela Ferry XDR-specific and XDR/MDR-specificmutations in a diverse set of M. tuberculosis MDR and XDR isolates as well as DS controls.Tugela Ferry mutations that were found to be present in other sequenced DS M. tuberculosisgenomes and mutations that were already known to confer resistance to individual antibioticswere excluded, as our interest was to discover new resistance-associated mutations. We alsoexcluded six mutations in the PPE/PE-PGRS gene family and the other repetitive genes,because these genes are known to be hyper-variable even in DS M. tuberculosis and they wouldbe unlikely to have a relationship to drug resistance [13]. Thus, our final study focused on fivesynonymous, five non-synonymous and two non-coding mutations in seven genes/regions withknown functions and five hypothetical genes/regions (Fig. 1).

To ensure that mutations were studied in a highly diverse set of M. tuberculosis strains, weselected five or six drug-resistant and five DS M. tuberculosis isolates from each of the fourmajor M. tuberculosis phylogenetic groups, and two or three drug-resistant and two or threeDS isolates from each of the five phylogenetic sub-groups as described previously [12] (Fig.2). Thirty-one of the 35 drug-resistant isolates were MDR, three were resistant to at least twodrugs and one was mono-resistant (Table S1). Six XDR isolates from phylogenetic SNP clustergroup (SCG) 2 and SCG 3a isolated from two different geographic locations (Korea andUganda) were also analyzed giving a total of 41 drug-resistant and 34 drug susceptible strains.

We did not find any of the 12 Tugela Ferry XDR-specific or XDR/MDR-specific mutations inany of the 41 drug-resistant isolates in our study (Table 1). The complete absence of any ofthese mutations in our widely representative sample of drug-resistant isolates strongly suggeststhat these mutations do not have broad biological relevance in the evolution of XDRtuberculosis. We also considered the possibility that the mutations observed in the Tugela FerryXDR strain might identify new gene regions rather than specific new mutations that playimportant roles in drug-resistance. To this end, we sequenced approximately 600 bp of DNAflanking each of the 12 mutations in the 41 drug-resistant isolates. We also sequenced the entire1357bp Rv3616c-Rv3617 intergenic region. The DNA sequencing revealed 26 new mutationsin five genes or intergenic regions (Rv0103c, Rv2000, intergenic-Rv3616c-Rv3617, Rv3806c



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and Rv3921c) (Table 2). Nine of these mutations were present in more than one drug-resistantisolate, while 13 mutations were identified in only one drug-resistant isolate. The same regionswere then sequenced in the 34 phylogenetically-matched drug-susceptible M. tuberculosisisolates (Table 2). We found that eight of the nine mutations previously identified in more thanone drug-resistant isolate were also present in at least one drug-susceptible isolate, stronglysuggesting that these mutations were not associated with drug resistance. Thus, only the −992G to T mutation in the Rv3616c-Rv3617 intergenic region was present in more than one drug-resistant isolate but not present in any drug-susceptible isolate; however this mutation waspresent at a 2/40 (5%) frequency and occurred in isolates from the same SCG as describedbelow. Three new mutations which were only present in the drug susceptible isolates were alsoidentified. Of these, one occurred in more than one drug-susceptible isolate.

Commonly occurring drug-resistance associated mutations have a distinct phylogeneticdistribution, suggestive of convergent evolution, compared to neutral mutations in M.tuberculosis. Common resistance-associated mutations can be shown to arise independentlymultiple times in broadly representative tuberculosis populations and cannot be traced back toa single common ancestor in a phylogenetic analysis. Mutations that are not strongly selectedwill be monophyletic and appear to have arisen only once in this population [8]. We performeda phylogenetic analysis of each of the 11 mutations which were found in more than one M.tuberculosis isolate, examining their distribution within the context of the entire 75 isolatestudy set (Fig. 3a to 3d). Each mutation either mapped to a single phylogenetic branch ormapped to adjoining branches. This included the −992 G to T mutation in the Rv3616c-Rv3617intergenic region which was confined to SCG 1 (Fig. 3b). With one exception, when a mutationwas present on adjoining branches, at least one of the branches was 100% mutant suggestinga single mutational event in a common ancestor. These results provide strong evidence that thenew mutations discovered in the drug resistant isolates are associated with strain phylogenyrather than drug resistance. The lone exception occurred with the −706 T to C mutation in theRv3616c - Rv3617 intergenic region. This mutation, which was present in both MDR and DSisolates was found in 50% of SCG 1 isolates as well as one isolate on the contiguous SCG 3abranch. The absence of a complete monophyletic distribution leaves open the possibility thatthis mutation has multiple ancestries and developed under selective pressure. However, thelack of any association to drug resistance, its relative paucity in the study set and its completeabsence on most of the phylogeny suggest that this is not an important fitness mutation.

Eleven of the 33 Tugela Ferry MDR-XDR and XDR mutations had been previously associatedwith drug resistance. Two of these mutations at katG315 and inhA −15 which are known tocause resistance to the antituberculosis drug isoniazid were examined in our 75 drug-resistantand DS strain set. These known resistance-associated mutations were well represented amongthe drug-resistant isolates, but were not present in any of the DS isolates. The phylogeneticdistribution of the katG315 and inhA −15 mutations also contrasted markedly with thedistribution of the mutations that were newly identified in this study (Fig. 3e and 3f). Both thekatG and the inhA mutations were distributed on multiple phylogenetic branches, consistentwith previous studies [8]. The marked contrast in the distribution of established drug resistancemutations compared to the mutations newly discovered here provides further evidence that thenew mutations are not associated with drug resistance or a significant fitness advantage.

We also sequenced the entire length of each gene identified by the Tugela Ferry MDR-XDRmutations in our six XDR isolates. Five additional mutations in Rv0020c, Rv0663, Rv1145and Rv2000 were identified. Four mutations were present in multiple isolates and one mutationwas only found in a single isolate. We examined our DS isolates matched by phylogeneticgroup for each of these mutations. All four of the mutations present in more than one XDRisolate were also found in one or more DS isolates belonging to the same SCGs as the XDRisolates (Table 3).



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One of the mutations which we observed most frequently was located at position −41 (G to T)of the Rv3616c - Rv3617 intergenic region. This mutation was of interest even though it waspresent in both susceptible and resistant isolates because the Rv3612c-Rv3616c operon appearsto be required for the secretion of ESAT-6 and CFP-10 which are vital to M. tuberculosisvirulence [14], because the mutation was present in all of the highly virulent SCG2-Beijingclade isolates, and because the −41 mutation was in close proximity to the −55 mutationdescribed in the Tulega Ferrry XDR isolate. These observations prompted us to investigate ifthis mutation had any effect on ESAT-6 secretion. However, we did not find any difference inthe transcription of Rv3616c or the extra-cellular secretion of ESAT-6 using RT-PCR andwestern blot analysis, respectively in either −41 G to T mutant or wild type isolates (data notshown).

DiscussionThis study represents the first comprehensive analysis, to our knowledge, of XDR and MDR-XDR mutations discovered by the Broad Institute KZN XDR sequencing project. It suggeststhat XDR tuberculosis can evolve in a virulent form without generalizable fitness changes orother XDR-specific mutations. The Broad Institute sequencing effort provided a uniqueopportunity to determine whether secondary mutations that predispose to MDR and/orcompensate for the fitness costs of resistance-conferring mutations develop in tandem withXDR evolution. We were unable to identify any mutations that the Tugela Ferry XDR strainshared with unrelated drug-resistant M. tuberculosis isolates other than loci already known tobe associated with drug-resistance. Thus, the Tugela Ferry genome sequence does not appearto provide new generalizable insights into the evolution of XDR tuberculosis. Furthermore,our examination of the gene and gene region surrounding the MDR-XDR and XDR mutationsdid not reveal any additional candidates for secondary resistance or fitness mutations. However,it was not possible to completely rule out either of these functions for mutations which wereonly detected one time in the study set.

It remains possible that the Tugela Ferry strain developed an unusual set of adaptations to XDRthat were not present in any of the other drug-resistant strains tested. However, both heightenedtransmission and high mortality have been repeatedly described when immunocompromisedpatients are exposed to drug-resistant M. tuberculosis. Therefore, it is more likely that theTugela Ferry strain does not contain unique genetic adaptations to XDR. Rather, the widespreaddistribution and high mortality of this strain can probably be attributed to the health status ofthe host and possibly, the quality and availability of medical treatment. Our results do notcompletely rule out the possibility that drug-resistant M. tuberculosis develops mutations thatsupport MDR/XDR acquisition and maintain fitness. Selection for mutations of this type maybe dictated by treatment and/or host factors that were not widespread in the Tugela Ferry XDRoutbreak. For example, fitness mutations may not be required to maintain full virulence inimmunocompromised hosts. Furthermore, it is possible that certain mutations in knownresistance-associated genes can provide drug-resistance without attenuation (such as katG315mutations [15]) or can predispose to resistance developing across drug classes (such asmutations in embB306 [9,16]). Both of these mutations were found in the Tugela Ferry genome,and these already established mutations rather than the new mutations of unknown functionmay fully explain the ability of this strain to be transmitted and cause disease. It will requirefurther genome comparisons of additional XDR isolates, particularly from HIV negativepatients to resolve these questions.

Supplementary MaterialRefer to Web version on PubMed Central for supplementary material.



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AcknowledgmentsThis work was partially supported by NIH grant AI065663. The funders had no role in study design, data collectionand analysis, decision to publish, or preparation of the manuscript.

References1. LoBue P. Extensively drug-resistant tuberculosis. Curr Opin Infect Dis 2009;22:167–173. [PubMed:

19283912]2. Gandhi NR, Moll A, Sturm AW, et al. Extensively drug-resistant tuberculosis as a cause of death in

patients co-infected with tuberculosis and HIV in a rural area of South Africa. Lancet 2006;368:1575–1580. [PubMed: 17084757]

3. Ramaswamy S, Musser JM. Molecular genetic basis of antimicrobial agent resistance inMycobacterium tuberculosis: 1998 update. Tuber Lung Dis 1998;79:3–29. [PubMed: 10645439]

4. Gagneux S. Fitness cost of drug resistance in Mycobacterium tuberculosis. Clin Microbiol Infect2009;5:66–68. [PubMed: 19220360]

5. Gagneux S, Long CD, Small PM, Van T, Schoolnik GK, Bohannan BJ. The competitive cost ofantibiotic resistance in Mycobacterium tuberculosis. Science 2006;312:1944–1946. [PubMed:16809538]

6. Koenig R. Tuberculosis. Few mutations divide some drug-resistant TB strains. Science 2007;318:901–902. [PubMed: 17991835]

7. Broad Institute TB Website.www.broad.mit.edu/annotation/genome/mycobacterium_tuberculosis_spp/ToolsIndex.html

8. Hazbon MH, Motiwala AS, Cavatore M, Brimacombe M, Whittam TS, Alland D. Convergentevolutionary analysis identifies significant mutations in drug resistance targets of Mycobacteriumtuberculosis. Antimicrob Agents Chemother 2008;52:3369–3376. [PubMed: 18591265]

9. Hazbon MH, Bobadilla del Valle M, Guerrero MI, et al. Role of embB codon 306 mutations inMycobacterium tuberculosis revisited: a novel association with broad drug resistance and IS6110clustering rather than ethambutol resistance. Antimicrob Agents Chemother 2005;49:3794–3802.[PubMed: 16127055]

10. Hazbon MH, Brimacombe M, Bobadilla del Valle M, et al. Population genetics study of isoniazidresistance mutations and evolution of multidrug-resistant Mycobacterium tuberculosis. AntimicrobAgents Chemother 2006;50:2640–2649. [PubMed: 16870753]

11. Alland D, Lacher DW, Hazbon MH, et al. Role of large sequence polymorphisms (LSPs) in generatinggenomic diversity among clinical isolates of Mycobacterium tuberculosis and the utility of LSPs inphylogenetic analysis. J Clin Microbiol 2007;45:39–46. [PubMed: 17079498]

12. Filliol I, Motiwala AS, Cavatore M, et al. Global phylogeny of Mycobacterium tuberculosis basedon single nucleotide polymorphism (SNP) analysis: insights into tuberculosis evolution, phylogeneticaccuracy of other DNA fingerprinting systems, and recommendations for a minimal standard SNPset. J Bacteriol 2006;188:759–772. [PubMed: 16385065]

13. Cole ST. Comparative and functional genomics of the Mycobacterium tuberculosis complex.Microbiology 2002;148:2919–2928. [PubMed: 12368425]

14. Raghavan S, Manzanillo P, Chan K, Dovey C, Cox JS. Secreted transcription factor controlsMycobacterium tuberculosis virulence. Nature 2008;454:717–721. [PubMed: 18685700]

15. Pym AS, Saint-Joanis B, Cole ST. Effect of katG mutations on the virulence of Mycobacteriumtuberculosis and the implication for transmission in humans. Infect Immun 2002;70:4955–4960.[PubMed: 12183541]

16. Safi H, Sayers B, Hazbon MH, Alland D. Transfer of embB codon 306 mutations into clinicalMycobacterium tuberculosis strains alters susceptibility to ethambutol, isoniazid, and rifampin.Antimicrob Agents Chemother 2008;52:2027–2034. [PubMed: 18378710]



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http://www.broad.mit.edu/annotation/genome/mycobacterium_tuberculosis_spp/ToolsIndex.html

Figure 1. Flow chart for selection of genes included in the study



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Figure 2. Phylogenetic locations of the study isolatesEach branch shows the location of SNP cluster groups (SCGs), including subgroups as definedby an analysis of 212 SNP markers on 324 M. tuberculosis isolates [12]. Study isolates wereplaced on the phylogenetic tree using 9 SNP markers identified for this purpose [11]. Thepositions of the M. tuberculosis reference strains 210, CDC1551, and H37Rv and of M.bovis strain AF2122/97 are indicated. Positions of the drug-susceptible (DS), multi-drugresistant (MDR) and extensively-drug resistant (XDR) are also indicated. The number of drug-resistant including MDR (R), pan-susceptible (S) and extensively drug-resistant (X) isolatesfrom each SCG are indicated.



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Figure 3.Panels A–D Phylogenetic distribution of the new mutations identified in the drug-resistant and susceptible isolates. The number of isolates located on each branch of thephylogenetic tree shown in Fig. 2 (and the % of isolates on that branch which contain thatmutation) is shown according to the indicated color code. Relevant linked SNP cluster groupsare highlighted in dark color. Nucleotide positions indicated are relative to the start site ofrespective genes. Panels E and F: Phylogenetic distribution of the inhA promoter andkatG315 mutations. The locations of each of the 75 study isolates on the phylogenetic treeshown in Fig. 2 that contained either the katG315 or the inhA promoter region SNPs are shown



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according to the indicated color code. Nucleotide positions indicated are relative to the startsite of respective genes.



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Table 1

Frequency of the Tugela Ferry-strain XDR and XDR-MDR specific mutations in a diverse sample of 41 drug-resistant M. tuberculosis isolates.

H37Rv gene H37Rv location Mutation # Mutation type

Rv0020c 24125 C/T 0 S (L440L)

Rv0103c 122107 G/A 0 NS (G23S)

Rv0663 756757 C/T 0 S (Y207Y)

Rv1145 1272321 C/A 0 NS (L25I)

Rv2000 2246032 T/C 0 NS (L275P)

Rv2141c 2401402 C/A 0 S (G109G)

Rv3459c 3879441 C/G 0 S (A84A)

Rv3471c 3889150 C/A 0 NS (D64E)

Rv3806c 4269271 T/C 0 NS (V188A)

Rv3921c 4409995 C/T 0 S (Y25Y)

Rv0571c and Rv0572cInt 664929 C/A 0 Intergenic

Rv3616c and Rv3617Int 4056430 T/C 0 Intergenic

#: Number of mutants

Int: Intergenic

NS: Non-synonymous

S: Synonymous


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Tabl

e 2

Mut

atio

ns id

entif

ied

in st

udy

isol

ates

by

sequ

enci

ng a

ppro

xim

atel

y 60

0 bp

flan

king

the

Tuge

la F

erry

-stra

in X

DR

and

XD

R-M

DR

mut

atio

n lo

ci.

H37

Rv

gene

aM

utat

ion

Res

ista

nt*

Susc

eptib

leM

utat

ion

type

n#

%n

#%

−4, G

to T

411

233

0-

Inte

rgen

ic

Rv0

103c

−42,

T to

C41

12

330

-In

terg

enic

−119

, G to

A41

12

330

-In

terg

enic

Rv2

000

656,

C to

A41

1024

346

18N

S (P

219Q

)

708,

T to

G41

25

341

3S

(A23

6A)

−41,

G to

Tb

4018

4533

1030

Inte

rgen

ic

−106

, del

Ab

401

333

0-

Inte

rgen

ic

−260

, A to

Gb

401

333

0-

Inte

rgen

ic

−318

, C to

Ab

4010

2533

515

Inte

rgen

ic

Rv3

616c

-Rv3

617In

t−6

61, T

to C

b41

25

322

6In

terg

enic

−706

, T to

Cb

414

1032

26

Inte

rgen

ic

−836

, G to

Ab

411

232

0-

Inte

rgen

ic

−847

, C to

Tb

410

032

13

Inte

rgen

ic

−992

, G to

Tb

402

530

0-

Inte

rgen

ic

228,

T to

C41

717

323

9S

(R76

R)

Rv3

806c

447,

A to

C41

717

345

15N

S (E

149D

)

517,

T to

G41

12

340

-N

S (S

173P

)

521,

A to

C41

12

340

-N

S (K

174T

)

536,

T to

G41

12

340

-N

S (I

179S

)

713,

T to

C41

12

340

-N

S (F

238S

)

864,

G to

A41

12

340

-S

(A28

8A)


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H37

Rv

gene

aM

utat

ion

Res

ista

nt*

Susc

eptib

leM

utat

ion

type

n#

%n

#%

116,

C to

G41

00

332

6N

S (A

39G

)

289,

C to

T41

12

330

-S

(L97

L)

Rv3

921c

336,

G to

C41

12

330

-N

S (M

112I

)

410,

G to

T41

12

331

3N

S (G

137V

)

468,

G to

A41

00

331

3S

(P15

6P)

Tot

al n

o. o

f iso

late

s41

34

* Incl

udes

6 X

DR

isol

ates

n: N

umbe

r of i

sola

tes s

eque

nced

#: N

umbe

r of m

utan

ts

Int:

Inte

rgen

ic re

gion

NS:

Non−s

ynon

ymou

s

S: S

ynon

ymou

s

a No

mut

atio

ns fo

und

in th

e 60

0bp

flank

ing

regi

on o

f Rv0

020c

, Rv0

571c

–Rv0

572c

Int ,

Rv0

663,

Rv1

145,

Rv2

141c

, Rv3

459c

, Rv3

471c

b Posi

tion

is g

iven

in re

fere

nce

to th

e tra

nsla

tiona

l sta

rt si

te o

f Rv3

616c


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Tabl

e 3

Add

ition

al m

utat

ions

iden

tifie

d in

XD

R st

udy

isol

ates

and

thei

r pre

senc

e in

mat

ched

DS

cont

rols

.

H37

Rv

gene

Mut

atio

nX

DR

Susc

eptib

leM

utat

ion

type

n#

%n

#%

Rv0

020c

707-

24, 1

8bp

del

66

100

66

100

6 am

ino

acid

del

etio

n

Rv0

663

1003

, C to

A6

583

63

50N

S (R

335S

)

1046

, A to

G6

610

06

583

NS

(D34

9G)

Rv1

145

711,

G to

A6

117

70

-S

(L23

7L)

829,

Ins A

66

100

76

86Fr

ame

shift

Tot

al n

o. o

f iso

late

s6

8

XD

R: E

xten

sive

ly d

rug

resi

stan

t

n: N

umbe

r of i

sola

tes s

eque

nced

#: N

umbe

r of m

utan

ts

NS:

Non

-syn

onym

ous

S: S

ynon

ymou

s


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Mutations in Extensively Drug‐Resistant Mycobacterium tuberculosis That Do Not Code for Known Drug‐Resistance Mechanisms